Camptothecin, a plant alkaloid isolated from trees indigenous to China, and analogues thereof such as 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 10,11-methylenedioxycamptothecin, 9-nitro-10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, 9-amino-10,11-methylenedioxycamptothecin, 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, DX-8951, GG211, 7-trimethylsilylmethylcamptothecin, and other analogues (collectively referred to herein as camptothecin drugs) are presently under study worldwide in research laboratories and cancer clinics. In lab tests and in clinical trials, these camptothecin drugs have aroused considerable interest as a result of their ability to halt the growth of a wide range of human tumors. For example, these drugs exhibit unprecedented high levels of antitumor activities against human colon cancer [Giovanella, et al. Science 246: 1046-1048 (Wash. D.C.) (1989)]. Camptothecin has also been shown to be effective against other experimental cancer types such as lung, breast, and malignant melanoma.
Camptothecin is thought to inhibit the proliferation of cancer cells by interfering with the breakage/reunion reaction of the enzyme topoisomerase I, a nuclear enzyme implicated in DNA replication and RNA transcription. A camptothecin drug stabilizes and forms a reversible enzyme-camptothecin-DNA ternary complex, designated the cleavage complex. The formation of the cleavable complex specifically prevents the reunion step of the breakage/union cycle of the topoisomerase reaction.
However, the clinical use of the camptothecin drugs is limited by their chemical properties. First, the camptothecin drugs are insoluble in water which hinders the delivery of the drug to the cancer cells. Second, the camptothecin drugs are extremely susceptible to hydrolysis; in an aqueous environment such as blood plasma, the half life is about 16 to 20 minutes and active lactone concentrations can fall to almost negligible levels depending upon the analogue. The camptothecin drugs each contain a substituted quinoline nucleus (rings A-C) and, at the opposite end an α-hydroxy lactone ring which is very unstable in aqueous environments. In blood plasma the ring is quickly opened to create the carboxylate form of the drug, which is poorly accumulated by cancer cells. Once internalized by the cancer cells, the carboxylate form exhibits no activity against its molecular target, topoisomerase I. Thus, the hydrolyzed product is ineffective at treating cancer. Moreover, the hydrolyzed product appears to be more toxic to healthy tissue than the camptothecin drugs.
Numerous attempts have, of course, been made to address these problems and shortcomings. To date, one of the most successful ways for maintaining camptothecin drugs in the antitumor-active lactone form is by stabilization of the drug in macromolecular lipid or surfactant assemblies such as liposomes or micelles, The liposomal or micellular stabilization of camptothecins is described in detail in U.S. Pat. Nos. 5,552,156 and 5,736,156 both to Burke. Specifically, the lactone ring of the camptothecin drugs is stabilized in the membrane of the vesicles. Typically, the camptothecin drugs bind the lipid bilayer membrane of the liposome and the surfactant monolayer membrane of the micelles. The liposome-bound or micelle-bound drug is protected from hydrolysis, thus preserving the antitumor activity of the drug.
The liposome is comprised of lipids such as, for example, phospholipids or cholesterol. For the camptothecin drugs which have a lower affinity for the liposome membrane and thus disassociate from the liposomal membrane to reside in the interior of liposome, the pH of the internal environment of the liposome is reduced thereby preventing hydrolysis of the camptothecin drug. Camptothecin drugs are also stabilized by association with micelles comprised of surfactants such as sodium dodecylsulfate (SDS), octylphenolpolyoxyethylene glycol and sorbitan mono-oleate.
Recently the effectiveness of liposomal topotecan formulations were evaluated in animal models. Although topotecan exhibits good clinical activity against a variety of tumor types, it undergoes rapid hydrolysis in vivo from the active, lactone species to the inactive carboxylate. This inactivation, coupled with cell-cycle-specific behavior and fairly rapid elimination, results in highly schedule-dependent clinical activity. Liposomal carriers accumulate selectively at tumor sites and can potentially act as sustained release systems maintaining therapeutic drug levels over a prolonged period. In the case of topotecan, such carriers might also protect the drug in its lactone form until released. A procedure whereby topotecan could be efficiently encapsulated within lipid-based carriers employing an ion gradient loading procedure and delivered to tumors has been developed (Madden et al., 1998). In contrast to earlier studies on liposomal delivery in which camptothecins were inserted predominantly into the lipid bilayer, the current technique entraps topotecan in the aqueous core of the carrier. Drug uptake was rapid (approximately 60 min. at 60° C.) and high encapsulation efficiencies (>95%) were readily achieved at relatively high drug:lipid ratios (0.1:1 mole ratio). Plasma elimination rates were compared for free topotecan and encapsulated drug (liposomal topotecan) in mice. Following a bolus intravenous injection (lateral tail vein) at 10 mg/kg, plasma level were followed for up to 24 hours. Plasma levels of topotecan given in the liposomal formulation were approximately two orders of magnitude higher than for free drug over this timecourse. Further, by employing carriers with an acidic interior the entrapped topotecan was protected as the lactone species (85% lactone at 24 hours). Antitumor efficacy was compared for free and liposomal topotecan in the murine L1210 model (i.p. tumor inoculation/i.v. treatment) using either a single bolus injection on day 1 (10 or 20 mg/kg), or bolus injections on days 1, 5, 9 (4 or 8 mg/kg). In all treatment schedules liposomal topotecan exhibited much greater antitumor activity than did free drug.
While the liposomal and micellar stabilization of camptothecin drugs described in U.S. Pat. Nos. 5,552,156 and 5,736,176 to Burke address and effectively overcome the instability and insolubility problems of camptothecin drugs administered in their free form, further improvements leading to more efficient, diversified and effective administration and treatment of tumors and various cancer types are still possible and are actively being sought.
Toward that end, we have developed the present invention. Specifically, we have found that direct noncovalent binding interactions of camptothecin drugs with oligonucleotides conserve the active forms of the drugs (i.e. the active lactone forms of camptothecin drugs are found to be stable when complexed with oligonucleotides). The oligonucleotides include single-stranded DNA, double-stranded DNA, antisense DNA, RNA, and catalytic RNA. The invention describes macromolecular assemblies which include both viral or non-viral oligonucleotide vectors. The viral gene delivery systems include retroviruses, adenoviruses, adeno-associated viruses, Herpes viruses, Vaccinia viruses, and other virus particles. The non-viral delivery systems include transfection vehicles, naked DNA for injection, gene gun particles, liposomes including cationic liposomes, virosomes, receptor-mediated delivery vehicles, and biodegradable and non-biodegradable polymer matrixes. The macromolecular assemblies can consist of the following materials: biodegradable and non-biodegradable polymers, lipids, carbohydrates, proteins and biologically relevant molecules which facilitate the delivery, accumulation and processing of the oligonucleotides and drugs to target tissues within the human body. Each of the above oligonucleotide delivery vectors contain oligonucleotides which act to stabilize the lactone forms of camptothecin drugs. The oligonucleotide contained within the vectors can serve as a stabilizing matrix for the drug and the stabilization can be effective over a wide pH range, the vectors also provide a means for the controlled, targeted and stable delivery of camptothecin drug to target tissue. In addition to stabilizing camptothecin, the oligonucleotides carried in the vectors serve an additional role in exerting gene therapy or pharmacological effects in general which can be envisioned to augment the effects of camptothecins on the host target tissue and the patient receiving the therapy. The combination of stable camptothecin drug delivery and gene therapy can have potentially unique and desirable consequences on inhibiting the spread of cancer and other disease states in humans and animals.